Epstein-Barr virus (EBV) is commonly present in humans. Antibodies to EBV polypeptides can be found in over 80% of human serum samples from the United States, and in even higher percentages from populations in Asian and African countries. In spite of its universal dominion, the consequences of EBV infection vary for different populations. Thus, for example, EBV is responsible for infectious mononucleosis (IM) in Western countries, and is implicated in Burkitt's lymphoma in Africa and nasopharyngeal carcinoma (NPC) in Asia. NPC is a major form of cancer in southern China where incidence rates are as high as 100 per 100,000 per year. The occurrence of these three EBV-associated diseases is rare other than in their normally associated populations. The risk-factors for different outcomes of EBV infection are not clear, although malarial infection in Africa and diet and use of medicinal herbs in Asia are potential co-factors.
There is a substantial amount of literature describing the serological screening for antibodies to EBV nuclear antigens (EBNA), early antigens (EA), and viral capsid antigens (VCA) in normal individuals and in patients with EBV-related diseases. EBNA is a viral product that is expressed in all cells carrying EBV DNA. Two separate components of EBNA have been identified by serology (see Proc. Nat'l. Acad. Sci., USA, 1983, 80: 5665-5669 AND 7650-7652). The major EBNA polypeptide is synthesized from the EBV BamHI K-restriction fragment and appears to be involved in the replication of EBV DNA as a plasmid (Nature, 1985, 313: 812-815).
Early antigens (EA) in Raji cells superinfected with EBV were first described as a complex of viral proteins that were expressed during the first 8-12 hours following infection (Science, 1970, 169: 188-190). The EA antigen complex consists of a "restricted" component (EA-R) and a "diffuse" component (EA-D) identified by their differential sensitivity to methanol and acetone fixation, sensitivity to proteases, and their location in the infected cells (Int. J. Cancer, 1971, 8: 272-282). The EA complex is among the first viral antigens to be expressed in lytic infection. The presence of antibodies to EA is considered to be an indicator of active viral production .
Antibody quantitation is generally determined by applying serially diluted serum samples to fixed smears of cells expressing, for example, EBNA, VCA or EA. Reactivity is detected by indirect immunofluorescence. An enzyme-linked immunosorbent assay (ELISA) for antibodies to EBV antigens, as provided by the present invention, offers the potential for an automated, highly-sensitive alternative to indirect immunofluorescence which does not require the experience or judgment of an expert technician.
Many investigators have described ELISA methods which employ crude or partially purified extracts of chemically induced cells to detect antibodies to early antigens. See Proc. Natl. Acad. Sci., USA, 1983, 80: 7650-7652; J. Infect. Diseases, 1982, 146: 734-740; J. Immunol. Meth., 1983, 63: 171-185 and J. Immonol. Meth., 1984, 67: 225-233. The development of monoclonal antibodies to early antigens (EA) enabled protein purification by affinity chromatography. Recently, Luka et al (J. Immunol. Meth., 1984, 67: 145-156) described an ELISA using EA-D purified by affinity chromatography using R3 monoclonal antibody (J. Virol., 1983, 47: 193-201) against the 50-52 kdalton EA-D protein. These workers harvested approximately 100 ug of partially purified EA-D per packed ml (10.sup.9) of TPA (12-0-tetradecanolylphorbol 13-acetate) induced P3HR-1 cells. Such preparation of EA-D from tissue culture cells appears to be too expensive for use in mass clinical screenings.
A principal object of the invention is to provide a less expensive and otherwise more convenient source of EBV antigens for use in quantitating EBV antibodies. Other objects will also be evident from the ensuing description.